Inhibitors of papilloma virus

ABSTRACT

A compound of formula (I) or its enantiomers or diastereoisomers thereof:                    
     wherein: A,; X, W, R 1 , Y; R 3 ; and R 4  are as defined herein. 
     The compounds of the invention may be used as inhibitors of the papilloma virus E1-E2-DNA complex. The invention further provides a method of treating or preventing human papilloma virus infection.

RELATED APPLICATIONS

Benefit of U.S. Provisional Application Serial No. 60/256,706, filed onDec. 18, 2000 is hereby claimed and said Application in incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions and methods forthe treatment of papilloma virus (PV) infection, particularly humanpapilloma virus (HPV). In particular, the present invention providesnovel indane derivatives, pharmaceutical compositions containing suchderivatives and methods for using these compounds in the treatment ofpapilloma virus infection. More particularly, the present inventionprovides compounds, compositions and methods for inhibiting papillomavirus DNA replication by interfering with the E1-E2-DNA complex duringinitiation of DNA replication.

BACKGROUND OF THE INVENTION

Papillomaviruses are non-enveloped DNA viruses that inducehyperproliferative lesions of the epithelia. The papillomaviruses arewidespread in nature and have been identified in higher vertebrates.Viruses have been characterized, amongst others, from humans, cattle,rabbits, horses, and dogs. The first papillomavirus was described in1933 as cottontail rabbit papillomavirus (CRPV). Since then, thecottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) haveserved as experimental prototypes for studies on papillomaviruses. Mostanimal papillomaviruses are associated with purely epithelialproliferative lesions, and most lesions in animals are cutaneous. In thehuman there are more than 75 types of papillomavirus that have beenidentified and they have been catalogued by site of infection: cutaneousepithelium and mucosal epithelium (oral and genital mucosa). Thecutaneous-related diseases include flat warts, plantar warts, etc. Themucosal-related diseases include laryngeal papillomas and anogenitaldiseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed.Lippincott—Raven Pub., Philadelphia, N.Y.).

There are more than 25 HPV types that are implicated in anogenitaldiseases, these are grouped into “low risk” and “high risk” types. Thelow risk types include HPV type 6, type 11 and type 13 and induce mostlybenign lesions such as condyloma acuminata (genital warts) and low gradesquamous intraepithelial lesions (SIL). In the United States there are 5million people with genital warts of which 90% is attributed to HPV-6and HPV-11. About 90% of SIL is also caused by low risk types 6 and 11.The other 10% of SIL is caused by high risk HPVs.

The high risk types are associated with high grade SIL and cervicalcancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52,and 58. The progression from low-grade SIL to high-grade SIL is muchmore frequent for lesions that contain high risk HPV-16 and 18 ascompared to those that contain low risk HPV types. In addition, onlyfour HPV types are detected frequently in cervical cancer (types 16, 18,31 and 45). About 500,000 new cases of invasive cancer of the cervix arediagnosed annually worldwide (Fields, 1996, supra).

Treatments for genital warts include physical removal such ascryotherapy, CO₂ laser, electrosurgery, or surgical excision. Cytotoxicagents may also be used such as trichloroacetic acid (TCA), podophyllinor podofilox. Immunotherapy is also available such as Interferon orImiquimod. These treatments are not completely effective in eliminatingall viral particles and there is either a high cost incurred oruncomfortable side effects related thereto. In fact, there are currentlyno effective antiviral treatments for HPV infection since recurrentwarts are common with all current therapies (Beutner & Ferenczy, 1997,Amer. J. Med., 102(5A), 28-37).

The ineffectiveness of the current methods to treat HPV infections hasdemonstrated the need to identify new means to control or eliminate suchinfections. In recent years, efforts have been directed towards findingantiviral compounds, and especially compounds capable of interferingwith viral replication at the onset of infection (Hughes, 1993, NucleicAcids Res. 21:5817-5823).

The life cycle of PV is closely coupled to keratinocyte differentiation.Infection is believed to occur at a site of tissue disruption in thebasal epithelium. Unlike normal cells, the cellular DNA replicationmachinery is maintained as the cell undergoes vertical differentiation.As the infected cells undergo progressive differentiation the viralgenome copy number and viral gene expression in turn increase, with theeventual late gene expression and virion assembly in terminallydifferentiated keratinocytes and the release of viral particles (Fields,supra).

The coding strands for each of the papillomavirus contain approximatelyten designated translational open reading frames (ORFs) that have beenclassified as either early ORFs or late ORFs based on their location inthe genome. E1 to E8 are expressed early in the viral replication cycle,and two late genes (L1 and L2) encode the major and minor capsideproteins respectively. The E1 and E2 gene products function in viral DNAreplication, whereas E5, E6 and E7 are expressed in connection with hostcell proliferation. The L1 and L2 gene products are involved in virionstructure. The function of the E3, E4 and E8 gene products is uncertainat present.

Studies of HPV have shown that proteins E1 and E2 are both essential andsufficient for viral DNA replication in vitro (Kuo et al., 1994, J.Biol. Chem. 30:24058-24065). This requirement is similar to that ofbovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree ofsimilarity between E1 and E2 proteins and the ori-sequences of allpapillomaviruses (PV) regardless of the viral species and type (Kuo etal., 1994, supra).

Evidence emanating from studies of BPV-1 have shown that E1 possessesATPase and helicase activities that are required in the initiation ofviral DNA replication (Seo et al., 1993a, Proc. Natl. Acad. Sci. USA90:702-706; Yang et al., 1993, Proc. Natl. Acad. Sci. 90:5086-5090; andMacPherson et al., 1994, 204:403-408).

The E2 protein is a transcriptional activator that binds to E1 proteinand forms a complex that binds specifically to the ori sequence (Mohr etal., 1990, Science 250:1694-1699). It is believed that E2 enhancesbinding of E1 to the BPV origin of replication (Seo et al., 1993b, Proc.Natl. Acad. Sci., 90:2865-2869). In HPV, Lui et al. suggested that E2stabilizes E1 binding to the ori (1995, J. Biol. Chem.,270(45):27283-27291).

To thwart this disease, a chemical entity that would interfere withviral DNA replication is therefore desirable. The present inventiontherefore provides such compounds, compositions or methods that inhibitpapilloma viral replication. More particularly, the compounds andcomposition of the present invention interfere with the E1-E2-DNAcomplex during the viral replication cycle.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a compound of formula (I), orits enantiomers or diastereoisomers thereof:

wherein:

A is a 5- or 6-membered homocyclic ring, or a 5- or 6-memberedheterocyclic ring containing 1 or more heteroatoms selected from N, Oand S;

X is H and W is OH; or X and W together form a carbonyl group or anepoxide;

R¹ is H; or one or two substituents independently selected from thegroup consisting of: hydroxy; halo; lower alkyl; lower alkoxy; lowerthioalkyl; haloalkyl (e.g. trifluoromethyl); or —C(O)R² wherein R² islower alkyl, aryloxy or benzyloxy;

Y is phenyl optionally mono- or di-substituted with R⁵ or C(O)R⁶,wherein R⁵ is lower alkyl, lower cycloalkyl, lower alkoxy, halo,hydroxy, nitrile or trifluoromethyl, and R⁶ is lower alkyl, lowercycloalkyl, lower alkoxy, hydroxy or trifluoromethyl; said phenyl ringbeing optionally fused with a saturated or unsaturated 4 to 6-memberedring optionally containing a heteroatom selected from N, O and S;

or Y is a heterocycle (Het) containing one or more heteroatom selectedfrom N, O or S, said Het optionally mono- or di-substituted with R⁵ orC(O)R⁶, wherein R⁵ and R⁶ are as defined above; said Het beingoptionally fused with a saturated or unsaturated 4 to 6-membered ringoptionally containing a heteroatom selected from N, O and S;

or Y is ethylene-phenyl, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein said phenyl ring is optionallymono- or di-substituted with R⁵ or C(O)R⁶, wherein R⁵ and R⁶ are asdefined above; said phenyl ring being optionally fused with a saturatedor unsaturated 4- to 6-membered ring optionally containing a heteroatomselected from N, O and S;

or Y is ethylene-Het, said ethylene moiety being optionallymono-substituted with lower alkyl, wherein Het is optionally mono- ordi-substituted with R⁵ or C(O)R⁶, wherein R⁵ and R⁶ are as definedabove; said Het being optionally fused with a saturated or unsaturated 4to 6-membered ring optionally containing a heteroatom selected from N, Oand S;

R³ is selected from the group consisting of: lower alkyl, lowercycloalkyl, lower alkylene, aryl or lower aralkyl, all of whichoptionally mono- or di-substituted with:

lower alkyl, lower cycloalkyl, haloalkyl, halo, CN, azido, lower alkoxy,(lower alkyl)acyl, C₁₋₆ thioalkyl, C₁₋₆ alkylsulfonyl, NHC(O)-loweralkyl, NHC(O)-aryl, NHC(O)—O-lower alkyl, NHC(O)O-aryl, aryl, aryloxy,hydroxy, nitro, amino, or Het, said Het optionally mono- ordi-substituted with lower alkyl, lower cycloalkyl, lower alkoxy, halo,hydroxy, nitrile, trifluoromethyl, C(O)R⁶ wherein R⁶ is as definedabove;

said lower cycloalkyl, aryl, lower aralkyl or Het being optionally fusedwith a saturated or unsaturated 4 to 6-membered ring optionallycontaining a heteroatom selected from N, O and S; and

R⁴ is a carboxylic acid, a salt or an ester thereof;

and wherein wavy lines represent bonds of unspecified stereochemistry;

and with the provisos that:

(1) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is 4-methylphenyl, then R³ cannot be benzyl, 3-fluorophenyl,or 4-nitrophenyl;

(2) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and R³ is cyclohexyl, then Y cannot be 4-iodophenyl or4-methylphenyl;

(3) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is 4-fluorophenyl, then R³ cannot be4-ethyloxycarbonylphenyl;

(4) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is 2-methylphenyl then R³ cannot be 4-nitrophenyl;

(5) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is 2-methylphenyl, then R³ cannot be phenyl or2-bromo-4-methylphenyl;

(6) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is 4-chlorophenyl, then R³ cannot be 2-methoxyphenyl or1,3-benzodioxolyl;

(7) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is 4-ethylphenyl, then R³ cannot be 3-fluorophenyl; and

(8) when A is benzene, R¹ is hydrogen, X and W together form a carbonylgroup and Y is phenyl, then R³ cannot be phenyl.

Alternatively, the first aspect of the invention provides compoundshaving the following formulae, selected from the group consisting of:

wherein A, X, W, R¹, Y, R³ and R⁴ are as defined above, with theprovisos indicated above.

Compounds of the invention may also be represented by formula I in forms(2) and (3):

wherein A, X, W, R¹, Y and R³ are as defined above, with the provisosindicated above.

As will be recognized by persons skilled in the art, the compounds informs (2) and (3) are readily converted to compounds of formula (I) inform (1). Without wishing to be bound by theory, it is believed that thecompounds of formula (I) are in equilibrium between forms (1), (2) or(3) depending on the solvent and the pH in which they are dissolved. Ithas however been demonstrated that compounds of formula (I) arebiologically active in form (1), and that the compounds in forms (2) and(3) will hydrolyze in conditions reproducing mammalian plasma (pH 7.4),to yield biologically active form (1).

In a second aspect, the invention provides a pharmaceutical compositioncomprising an anti-papillomavirus virally effective amount of a compoundof formula (I) or a therapeutically acceptable salt or ester thereof, inadmixture with a pharmaceutically acceptable carrier medium or auxiliaryagent.

In a third aspect, the invention provides a method for treating apapillomavirus viral infection in a mammal by administering to themammal an anti-papilloma virus virally effective amount of the compoundof formula (I) or a therapeutically acceptable salt or ester thereof, ora composition as described above, (all without the provisos indicatedabove for formula (I).

In fourth aspect, the invention provides a method for inhibiting thereplication of papillomavirus by exposing virally infected cells to anamount of the compounds of formula (I) inhibiting the papilloma virusE1-E2-DNA complex, or a therapeutically acceptable salt or esterthereof, or a composition as described above, (all without the provisosindicated above for formula (I).

In a fifth aspect, the invention provides a use of compounds of formula(I) (without the provisos indicated above for formula (I)) for themanufacture of a medicament for treating a papillomavirus viralinfection,.

In an sixth aspect, the invention provides a method of preventingperinatal transmission of HPV from mother to baby, by administering acompound of formula (I) (without the provisos indicated above forformula (I)) to the mother prior to giving birth.

In a seventh aspect, the invention provides a use of compounds offormula (I) (without the provisos indicated above for formula (I)) forthe manufacture of a medicament for preventing perinatal transmission ofHPV from mother to baby prior to giving birth.

In an eighth aspect, the invention provides an intermediate compound offormula (vi):

wherein Y and R³ are as defined above, or enantiomers ordiastereoisomers thereof, with the provisos indicated above for formula(I).

In a ninth aspect, the invention provides an intermediate compound offormula (xx), said compound having trans/trans relative stereochemistry:

wherein Y, R³, and R⁴ are as defined above, with the provisos indicatedabove for formula (I).

In an tenth aspect, the invention provides an intermediate compound offormula (xxvi):

wherein R¹, R³ and Y are as defined above,

or a salt or an ester thereof, or enantiomers and diastereoisomersthereof, without the provisos indicated above for formula (I).

In a eleventh aspect, the invention provides an intermediate compound offormula (xxxii):

wherein R¹, R³ and Y are as defined above,

or a salt or an ester thereof, or enantiomers and diastereoisomersthereof, without the provisos indicated above for formula (I).

In a twelfth aspect, the invention provides a process for producingcompounds of formula I′,

wherein X, R¹, W, Y, R³, and R⁴ are as defined above, with the provisosindicated above for formula (I), comprising:

a) hydrolyzing, in a mixture of aqueous base and a co-solvent, eitherintermediate compound vi or intermediate compound xx

to produce compounds of formula I′, wherein R³, R⁴, and Y are as definedabove.

In a thirteenth aspect, the invention provides a process for producingcompounds of formula I″,

wherein X and W together form a carbonyl group, R⁴ is a carboxylic acidor an ester, and R¹, Y, and R³ are as defined above, without theprovisos indicated above for formula (I), comprising:

a) hydrolyzing, in a mixture of aqueous base and a co-solvent,intermediate compound xxvi,

so as to produce compounds of formula I″, wherein R¹, Y, and R³ are asdefined above.

In a fourteenth aspect, the invention provides, a process for producingcompounds of formula I′″,

wherein X and W together form a carbonyl group, R⁴ is a carboxylic acidor an ester, and R¹, Y, and R³ are as defined above, without theprovisos indicated above for formula (I), comprising:

a) hydrolyzing, in a mixture of aqueous base and a co-solvent,intermediate compound xxxii

so as to produce compounds of formula I′″, wherein R¹, Y, and R³ are asdefined above.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

As used herein, the following definitions apply unless otherwise noted:

The term “halo” as used herein means a halo radical selected from bromo,chloro, fluoro or iodo.

The term “lower alkyl” (or C₁₋₆ alkyl) as used herein, either alone orin combination with another radical, means straight or branched-chainalkyl radicals containing up to six carbon atoms and includes methyl,ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl,2-methylpropyl and 1,1-dimethylethyl. The term “C₀₋₆ alkyl” preceding aradical means that this radical can optionally be linked through a C₁₋₆alkyl radical or the alkyl may be absent (C₀).

The term “lower cycloalkyl” as used herein, either alone or incombination with another radical, means saturated cyclic hydrocarbonradicals containing from three to seven carbon atoms and includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The term “lower alkoxy” as used herein means straight chain alkoxyradicals containing one to four carbon atoms and branched chain alkoxyradicals containing three to four carbon atoms and includes methoxy,ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Thelatter radical is known commonly as tert-butoxy.

The term “haloalkyl” as used herein means alkyl radical containing oneto six carbon atoms wherein one or more hydrogen atom is replaced by ahalogen atom (e.g. trifluoromethyl).

The term “amino” as used herein means an amino radical of formula —NH₂.The term “lower alkylamino” as used herein means alkylamino radicalscontaining one to six carbon atoms and includes methylamino,propylamino, (1-methylethyl)amino and (2-methylbutyl)amino. The term“di(lower alkyl)amino” means an amino radical having two lower alkylsubstituents each of which contains one to six carbon atoms and includesdimethylamino, diethylamino, ethylmethylamino and the like.

The term “acyl” as used herein, either alone or in combination withanother radical, refers to groups —C(O)R, wherein R is lower alkyl orlower alkoxy.

The term “C₆ or C₁₀ aryl” as used herein, either alone or in combinationwith another radical, means either an aromatic monocyclic systemcontaining 6 carbon atoms or an aromatic cyclic system containing 10carbon atoms. For example, aryl includes phenyl or naphthalene.

The term “C₇₋₁₆ aralkyl” as used herein, either alone or in combinationwith another radical, means an aryl as defined above linked through analkyl group, wherein alkyl is as defined above containing from 1 to 6carbon atoms. Aralkyl includes for example benzyl, and butylphenyl.

The term “Het” as used herein means a monovalent radical derived byremoval of a hydrogen from a five- or six-membered, saturated orunsaturated heterocycle containing from one to three heteroatomsselected from nitrogen, oxygen and sulfur. Optionally, the heterocyclemay bear one or two substituents; for example, N-oxido, lower alkyl,(C₁₋₃)alkyl-phenyl, lower alkoxy, halo, amino or lower alkylamino. Againoptionally, the five- or six-membered heterocycle can be fused to asecond cycloalkyl, an aryl (e.g. phenyl) or another heterocycle.

Examples of suitable heterocycles and optionally substitutedheterocycles include morpholine, thiadiazole, quinoline,3,4-methylene-dioxyphenyl, benzothiazole, pyrrolidine, tetrahydrofuran,thiazolidine, pyrrole, 1H-imidazole, 1-methyl-1H-imidazole, pyrazole,furan, thiophene, oxazole, isoxazole, thiazole, 2-methylthiazole,2-aminothiazole, 2-(methylamino)-thiazole, piperidine,1-methylpiperidine, 1-methylpiperazine, 1,4-dioxane, pyridine, pyridineN-oxide, pyrimidine, 2,4-dihydroxypyrimidine, 2,4-dimethylpyrimidine,2,6-dimethylpyridine, 1-methyl-1H-tetrazole, 2-methyl-2H-tetrazole,benzoxazole and thiazolo[4,5-b]-pyridine.

The term “pharmaceutically acceptable carrier” as used herein means anon-toxic, generally inert vehicle for the active ingredient which doesnot adversely affect the ingredient.

The term “effective amount” means a predetermined antiviral amount ofthe antiviral agent, i.e. an amount of the agent sufficient to beeffective against the virus in vivo.

The compounds of formula (I) can be obtained in the form oftherapeutically acceptable salts. The term “pharmaceutically acceptablesalt” as used herein includes those derived from pharmaceuticallyacceptable bases. Examples of suitable bases include choline,ethanolamine and ethylenediamine. Na+, K+, and Ca++ salts are alsocontemplated to be within the scope of the invention (also seePharmaceutical salts, Birge, S. M. et al., J. Pharm. Sci. (1977), 66,1-19, incorporated herein by reference).

The term “pharmaceutically acceptable ester” as used herein, eitheralone or in combination with another radical, means esters of thecompound of formula (I) in which the carboxyl function is replaced by analkoxycarbonyl function:

in which the R moiety of the ester is selected from alkyl (e.g. methyl,ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g. methoxymethyl);alkoxyacyl (e.g. acetoxymethyl); aralkyl (e.g. benzyl); aryloxyalkyl(e.g. phenoxymethyl); aryl (e.g. phenyl), optionally substituted withhalogen, C₁₋₄ alkyl or C₁₋₄ alkoxy. Other suitable prodrug esters can befound in Design of prodrugs, Bundgaard, H. Ed. Elsevier (1985)incorporated herewith by reference. Such pharmaceutically acceptableesters are usually hydrolyzed in vivo when injected in a mammal andtransformed into the acid form of the compound of formula (I).

With regard to the esters described above, unless otherwise specified,any alkyl moiety present advantageously contains 1 to 16 carbon atoms,particularly 1 to 6 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group.

In particular the esters may be a C₁₋₆ alkyl ester, an unsubstitutedbenzyl ester or a benzyl ester substituted with at least one halogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, nitro or trifluoromethyl.

Preferred Embodiments

According to a first embodiment of this invention, preferably compoundsof the invention are those in which ring A is a benzene ring, asrepresented by the formula I′:

Wherein X, W, R¹, Y and R³ are as defined above, with the provisosindicated above for formula (I). The compounds of formula I′ exist informs (1), (2) and (3), as described for the compounds of formula I.

Alternatively preferably, compounds of this invention are those in whichring A is a five-membered ring containing a sulfur atom, as representedby the formulae I″and I′″:

Wherein X, W, R¹, Y and R³ are as defined above, without the provisosindicated above for formula (I). The compounds of formulae I″ and I′″exist in forms (1), (2) and (3), as described for the compounds offormula I.

Alternatively even more preferably, compounds of the invention have thefollowing formula:

wherein R³ and R⁵ are as defined above, without the provisos indicatedabove for formula (I).

The compounds of the present invention can be synthesized as racemicmixtures and then separated in their respective single diastereoisomers.All such diastereoisomers are contemplated within the scope of thepresent invention.

Preferably, such diastereoisomers include mixture of compounds with thefollowing relative stereochemistry between [Y & C(O)NH—R³] and[C(O)NH—R³ & R⁴]:

Formulas (Ia) and (Ib) both represent racemic mixtures of compounds withthe relative stereochemistry referred to as “cis/cis”.

Formulas (Ic) and (Id) both represent racemic mixtures of compounds withthe relative stereochemistry referred to as “trans/trans”.

Formulas (Ie) and (If) both represent racemic mixtures of compounds withthe relative stereochemistry referred to as “trans/cis”.

Formula (Ig) and (Ih) both represent racemic mixtures of compounds withthe relative stereochemistry referred to as “cis/trans”.

More preferably, such diastereoisomers include mixture of compounds withthe relative stereochemistry “cis/cis”:

Also preferred are diastereoisomers with the relative stereochemistry“trans/trans”:

Most preferably, compounds of formula (I), present in an “cis/cis”relative stereochemistry that can also be represented as follows:

Still most preferably, the invention comprises pure enantiomers ofcompounds of formula (Ia) or (Ib) with the relative stereochemistry“cis/cis”:

With respect to compounds of formulae I, I′, I″, I′″, Ia, Ib, Ic, Id,Ie, If, Ig, and Ih, preferably X is H and W is OH; or X and W form acarbonyl group. Most preferably, X and W form a carbonyl group.

With respect to compounds of the formulae I′, I″ and I′″ preferably A isphenyl or thiophene. Most preferably, A is thiophene.

With respect to compounds of the formulae I′, I″ and I′″ preferably R¹is H; or one or two substituents independently selected from the groupconsisting of: hydroxy; halo; lower alkyl; lower alkoxy; lowerthioalkyl; haloalkyl (e.g. trifluoromethyl); or —C(O)R² wherein R² islower alkyl, aryloxy or benzyloxy.

More preferably, R¹ is H, halo or C₁₄ alkyl.

Even more preferably, R¹ is H, fluoro or methyl.

Most preferably, R¹ is H or methyl.

Preferably, Y is phenyl optionally mono- or di-substituted with R⁵ orC(O)R⁶, wherein R⁵ is lower alkyl, lower cycloalkyl, lower alkoxy, halo,hydroxy, nitrile or trifluoromethyl, and R⁶ is lower alkyl, lowercycloalkyl, lower alkoxy, hydroxy or trifluoromethyl; said phenyl ringbeing optionally fused with a saturated or unsaturated 4 to 6-memberedring optionally containing a heteroatom selected from N, O and S; or Yis ethylene-phenyl, said ethylene moiety being optionallymono-substituted with lower alkyl, wherein said phenyl ring isoptionally mono- or di-substituted with R⁵ or C(O)R⁶, wherein R⁵ and R⁶are as defined above; said phenyl ring being optionally fused with asaturated or unsaturated 4- to 6-membered ring optionally containing aheteroatom selected from N, O and S.

More preferably, Y is naphthyl, CH═CH-phenyl, C(CH₃)═CH-phenyl orphenyl, wherein the phenyl ring is optionally mono- or di-substituted atthe 3, 4, or 5 position with R⁵, wherein R⁵ is halo, C₁₋₄ alkyl,hydroxy, CF₃ or NHC(O)-(lower alkyl).

Still more preferably, Y is phenyl optionally substituted with: 3,4-Cl;3-F,4-Cl; 3-Cl,4-F; 3,4-Br; 3-F,4-CH₃; 3,4-CH₃; 3-CF₃, NHC(O)—(CH₂)₃CH₃and

Most preferably, Y is phenyl optionally substituted with:

3,4-Cl and 3,4-Br.

Preferably, R³ is selected from the group consisting of:

cyclohexyl; C₁₋₆ alkyl; C₁₋₆ thioalkyl; (C₁₋₆ alkyl)phenyl wherein thephenyl ring is optionally substituted with:

lower alkyl, CF₃, halo, CN, azido, lower alkoxy, (lower alkyl)acyl, C₁₋₆thioalkyl, C₁₋₆ alkylsulfonyl, NHC(O)-lower alkyl, aryl, aryloxy,hydroxy, nitro, amino, or Het, said Het optionally mono- ordi-substituted with lower alkyl, lower alkoxy, halo, hydroxy, nitrile,trifluoromethyl;

Even more preferably, R³ is selected from the group consisting of: C₁₋₆alkyl; C₁₋₆ thioalkyl;

Most preferably, R³ is selected from the group consisting of:

Particularly preferred compounds of the invention are compounds havingthe formula I″. Of compounds having the formula I″, those having the“cis/cis” configuration are particularly preferred.

Preferably, R⁴ is a carboxylic acid, a salt or an ester thereof.

Different Forms of Compounds of Formula (I)

Compounds of formula (I) according to the invention can presentthemselves in different forms according to the solvent and the pH inwhich they are dissolved. For example, compound 1001 (Table 1, form 1)can exist in equilibrium with compound 2001 (see Table 2, hereinafter)and compound 3005 (Table 3) in form (3) when dissolved in phosphatebuffer at pH 7.4. Without wishing to be bound by theory, it is believedthat the predominant form in solution at pH 7.4 is represented by form(1).

Specific Embodiments

Included within the scope of this invention are all compounds of formulaformulae I, I′, I″, I′″, Ia, Ib, I,c, Id, Ie, If, Ig, or Ih, aspresented in Tables 1 to 10 (with the exception of those compoundsexcluded by provisos).

Anti-papilloma Virus Activity

The antiviral activity of the compounds of formula (I) can bedemonstrated by biochemical and biological procedures showing theinhibitory effect of the compounds on DNA replication.

Preferably, the compounds of formula (I) as described above areinhibitory against human papillomavirus (HPV). More preferably thecompounds are active against HPV low risk or high risk type. Even morepreferably, against low risk type HPV (i.e. type 6, type 11 and type 13,and especially HPV type 11). Alternatively, the high-risk type isselected from the group consisting of types 16, 18, 31, 33, 35, 45, 52,or 58, preferably, type 16). Most preferably, the compounds of theinvention are directed against HPV types 6 and 11, even most preferably,against HPV-11.

A biochemical procedure for demonstrating anti-papilloma virus activityfor the compounds of formula (I) is described in the exampleshereinafter. This particular assay determines the ability of a testcompound to inhibit the activity (IC₅₀) of HPV-11 DNA replication. Morespecifically, in the assay described herein, the inhibitory activity ofthe test compound is evaluated based on its ability to interfere withthe E1-E2-DNA origin of replication interaction, thereby inhibitinginitiation of viral DNA replication.

Methods for demonstrating the inhibitory effect of the compounds offormula (I) on papilloma viral replication involving in vitro assays aredescribed in Examples 11 to 15 herein.

When a compound of formula (I), or one of its therapeutically acceptablesalts, is employed as an antiviral agent, it may be administered orally,topically or systemically to mammals, e.g. humans, rabbits or mice,alone or in a vehicle comprising one or more pharmaceutically acceptablecarriers, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard biological practice.

Whether it be termed treatment or prophylaxis, a compound of formula (I)may also be used to prevent perinatal transmission of HPV from mother tobaby, by administration to the mother prior to giving birth. Morespecifically, a compound of formula (I) may be used to prevent laryngealpapillomatosis in the baby.

For oral administration, the compound or a therapeutically acceptablesalt thereof can be formulated in unit dosage forms such as capsules ortablets each containing a predetermined amount of the active ingredient,ranging from about 25 to 500 mg, in a pharmaceutically acceptablecarrier.

For topical administration, the compound may be formulated inpharmaceutically accepted vehicles containing 0.1 to 5 percent,preferably 0.5 to 5 percent, of the active agent. Such formulations canbe in the form of a solution, cream or lotion.

For parenteral administration, the compound of formula (I) may beadministered by either intravenous, subcutaneous or intramuscularinjection, in compositions with pharmaceutically acceptable vehicles orcarriers. For administration by injection, it is preferred to use thecompounds in solution in a sterile aqueous vehicle which may alsocontain other solutes such as buffers or preservatives as well assufficient quantities of pharmaceutically acceptable salts or of glucoseto make the solution isotonic.

Suitable vehicles or carriers for the above noted formulations aredescribed in standard pharmaceutical texts, e.g. in “Remington's TheScience and Practice of Pharmacy”, 19th ed., Mack Publishing Company,Easton, Pa., 1995, or in “Pharmaceutical Dosage Forms And Drugs DeliverySystems”, 6th ed., H. C. Ansel et al., Eds., Williams & Wilkins,Baltimore, Md., 1995.

The dosage of the compound will vary with the form of administration andthe particular active agent chosen. Furthermore, it will vary with theparticular host under treatment. Generally, treatment is initiated withsmall increments until the optimum effect under the circumstance isreached. In general, the compound of formula I is most desirablyadministered at a concentration level that will generally affordantivirally effective results without causing any harmful or deleteriousside effects.

For oral administration, the compound or a therapeutically acceptablesalt may be administered in the range of 10 to 200 mg per kilogram ofbody weight per day, with a preferred range of 25 to 150 mg perkilogram.

For topical application, the compound of formula (I) may be administeredin a suitable formulation to the infected area of the body e.g. theskin, the genitalia, in an amount sufficient to cover the infected area.The treatment may be repeated, for example, every four to six hoursuntil lesions heal.

For systemic administration, the compound of formula (I) may beadministered at a dosage of 10 mg to 150 mg per kilogram of body weightper day, although the aforementioned variations will occur. However, adosage level that is in the range of from about 10 mg to 100 mg perkilogram of body weight per day is most desirably employed in order toachieve effective results.

Although the formulations disclosed herein are indicated to be effectiveand relatively safe medications for treating papilloma viral infections,the possible concurrent administration of these formulations with othermedications or agents to obtain beneficial results is also contemplated.Such other medications or agents include TCA, podophyllin, podofilox,Interferon or Imiquimod.

In addition to the above-mentioned antiviral agents, the compoundsaccording to the invention may also be used post-cryotherapy orpost-surgery or in combination with any other treatment for physicallyremoving warts.

Methodology and Synthesis

The synthesis of compounds of formula I′ is illustrated in Scheme I. Theradicals Y, R³ and R⁴ are as defined previously:

A): Commercially available indan-1,3-dione (i) [or prepared according toknown literature procedure: D. R. Bukle, N. J. Morgan, J. W. Ross, H.Smith, B. A. Spicer; J. Med. Chem. 1973, 16, 1334-1339] is condensedwith aldehyde (ii) in a protic solvent (e.g. ethanol or propanol) in thepresence of a catalytic amount of an organic amine (e.g. piperidine) toform the benzylidene (iii).

B): Benzylidene (iii) is converted to the epoxide (iv) by base-catalyzedoxidation with hydrogen peroxide in a protic solvent (such as methanol).

C): Epoxide (iv) undergoes thermal 1,3-dipolar cycloaddition in thepresence of maleimide (v) at temperatures ranging from 80 to 100° C. ina solvent such as toluene or xylene (ref.: M. Y. Krysin, I. K. Anohina,L. P. Zalukaev; Khimiya Geterotsiklicheskikh Soedinenii, 1987, 11,1463-1466). Thus racemic “cis/cis” (vi) and racemic “cis/trans” (vii)are obtained after purification (crystallization, flash columnchromatography, or preparative HPLC). In general maleimides such as (v)are commercially available or alternatively can be easily prepared usingliterature procedures (e.g. P. Y. Reddy, S. Kondo, T. Toru, Y. Ueno; J.Org. Chem., 1997, 62, 2652-2654).

D): Racemic “cis/cis” compound (vi) is hydrolyzed to yield its openedcarboxylate form (viii) also as “cis/trans” racemic mixture. Hydrolysisis achieved under aqueous basic conditions, such as aqueous sodiumhydroxide and acetonitrile as a co-solvent.

Alternatively steps A) and B) can be carried out as a “one pot” reactionusing an appropriate solvent (e.g. propanol) as described in Scheme II:

A): Diazoindan-1,3-dione (ix) [prepared according to literatureprocedure: J. Chem. Soc. Chem. Commun., 1990, 652-653] was reacted withaldehyde (ii) and maleimide (v) in the presence of a catalytic amount ofrhodium(II) to give racemic “cis/cis” compound (vi).

B): The corresponding carboxylate (viii) is made following thehydrolysis procedure described in Scheme I, step D).

C) This step presents a further method for synthesizing compounds offormula I′ in an alternative closed form. Racemic “cis/cis” compound(vi) is first hydrolyzed using procedure described in Scheme I, step D,followed with treatment with acid using dilute aqueous HCl to producehydroxylactone (x) as racemic “cis/cis”.

C′) Alternatively the sodium salt intermediate (viii) is passed througha reverse phase column (HPLC) using a trifluoroacetic acid containingeluent to yield the hydroxylactone (x).

Compounds of formula I′ wherein X and W form an epoxide are synthesizedas illustrated in Scheme III:

A): Racemic “cis/cis” compound (vi) is converted to the desiredinhibitors via first hydrolysis under basic conditions, following byacidification and treatment with diazomethane. Compounds (xi), (xii) and(xiii) are separated from the mixture by flash chromatography or bypreparative HPLC.

Scheme IV illustrates a general method for the synthesis of compounds offormula I′ wherein X is H and W is hydroxy:

A): Reduction of racemic “cis/cis” compound (vi) is achieved using ahydride source (e.g. sodium borohydride) to give mixtures of themonohydroxy derivatives (xiv and xv) in addition to the hydroxy lactone(xvi) having the relative stereochemistry as shown.

B): After separation, racemic (xiv and xv) are hydrolyzed using the sameprocedure as in Scheme I, step D) to give racemic (xvii and xviii) afterpreparative separation.

Scheme V illustrates the method for synthesizing compounds of formula I′with the relative stereochemistry in trans/trans.

A): The amide (xix) obtained using literature procedure (ex: G. B.Villeneuve and T. H. Chan Tetrahedron Letters, 1997, 38,6484) is reactedwith epoxide (iv) in toluene under refluxing conditions to yield thecycloadduct ester (xx) as racemic trans/trans isomers.

B): Hydrolysis of the ester (xx) is done as described in Scheme I, stepD) to give the desired carboxylate (xxi) also as racemic trans/transisomers.

Compounds of formula I″ and of formula I′″, may be made in an analogousmanner to those of formula I′, except that instead of indan-1,3-dione asstarting material, compound xxii is used.

A): Compound(xxii) [prepared by homologation of commercially available5-methyl-2-thiophenecarboxaldehyde with malonic acid, followed byreduction of the exocyclic double bond with sodium amalgam, or hydrogenover palladium, followed by cyclization with oxalyl chloride/AlCl₃, orpolyphosphoric acid, followed by oxidation withCrO₃/t-butylhydroperoxide] is condensed with aldehyde (ii) in a proticsolvent (e.g. ethanol or propanol) in the presence of a catalytic amountof an organic amine (e.g. piperidine) to form the benzylidene (xxiii).

B): Benzylidene (xxiii) is converted to the epoxide (xxiv) bybase-catalyzed oxidation with hydrogen peroxide in a protic solvent(such as methanol).

C): Epoxide (xxiv) undergoes thermal 1,3-dipolar cycloaddition in thepresence of maleimide (v) at temperatures ranging from 80 to 100° C. ina solvent such as toluene or xylene (ref.: M. Y. Krysin, I. K. Anohina,L. P. Zalukaev; Khimiya Geterotsiklicheskikh Soedinenii, 1987, 11,1463-1466). Thus racemic “cis/cis” (xxvi) and racemic “cis/trans”(xxvii) are obtained after purification (crystallization, flash columnchromatography, or preparative HPLC). In general maleimides such as (v)are commercially available or alternatively can be easily prepared usingliterature procedures (e.g. P. Y. Reddy, S. Kondo, T. Toru, Y. Ueno; J.Org. Chem., 1997, 62, 2652-2654).

D): Racemic “cis/cis” compound (xxvi) is hydrolyzed to yield its openedcarboxylate form (xxviii) also as “cis/cis” racemic mixture. Hydrolysisis achieved under aqueous basic conditions, such as aqueous sodiumhydroxide and acetonitrile as a co-solvent.

An alternate route to compounds of the formula I″ is shown in SchemeVII.

A) Compound xxix [prepared by homologation of commercially available-5-methyl-2-thiophenecarboxaldehyde with malonic acid, followed byreduction of the exocyclic double bond with sodium amalgam, or hydrogenover palladium, followed by cyclization with oxalyl chloride/AlCl₃, orpolyphosphoric acid] is condensed with aldehyde (ii) in the presence ofa catalytic amount of an acid catalyst (e.g. p-toluene sulfonic acid) inbenzene or toluene, to form the benzylidene (xxx).

B) Benzylidene (xxx) is converted to the epoxide (xxxi) by oxidation(e.g. CrO₃/t-butylhydroperoxide)

C) Epoxide (xxxi) undergoes thermal 1,3-dipolar cycloaddition in thepresence of maleimide (v) at temperatures ranging from 80 to 100° C. ina solvent such as toluene or xylene (ref.: M. Y. Krysin, I. K. Anohina,L. P. Zalukaev; Khimiya Geterotsiklicheskikh Soedinenii, 1987, 11,1463-1466). Thus racemic “cis/cis” (xxxii) and racemic “cis/trans”(xxxiii) are obtained after purification (crystallization, flash columnchromatography, or preparative HPLC). In general maleimides such as (v)are commercially available or alternatively can be easily prepared usingliterature procedures (e.g. P. Y. Reddy, S. Kondo, T. Toru, Y. Ueno; J.Org. Chem., 1997, 62, 2652-2654).

D) Racemic “cis/cis” compound (xxxii) is hydrolyzed to yield its openedcarboxylate form (xxxiv) also as “cis/cis” racemic mixture. Hydrolysisis achieved under aqueous basic conditions, such as aqueous sodiumhydroxide and acetonitrile as a co-solvent.

EXAMPLES

The present invention is illustrated in further detail by the followingnon-limiting examples. All reactions were performed in a nitrogen orargon atmosphere. Temperatures are given in degrees Celsius. Solutionpercentages or ratios express a volume to volume relationship, unlessstated otherwise.

Abbreviations or symbols used herein include:

DEAD: diethyl azodicarboxylate;

DIEA: diisopropylethylamine;

DMAP: 4-(dimethylamino)pyridine;

DMSO: dimethylsulfoxide;

DMF: dimethylformamide;

ES MS: electron spray mass spectrometry;

Et: ethyl;

EtOAc: ethyl acetate;

Et₂O: diethyl ether;

HPLC: high performance liquid chromatography;

iPr: isopropyl

Me: methyl;

MeOH: methanol;

MeCN: acetonitrile;

Ph: phenyl;

TBE: tris-borate-EDTA;

TBTU: 2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran;

MS (FAB) or FAB/MS: fast atom bombardment mass spectrometry;

HRMS: high resolution mass spectrometry;

Example 1 Preparation of Compounds 2013 (Table 2) and 1002 (Table 1)

Step a

To a solution of indan-1,3-dione (1a) (960 mg, 6.6 mmol) in EtOH (8.2mL) was added 3,4-dichlorobenzaldehyde (1b) (1.3 g, 7.2 mmol) followedby piperidine (3 drops). The reaction mixture was heated to reflux for30 min. After cooling, the reaction was diluted with EtOH (8 mL) and theprecipitate was filtered. The resulting solid was triturated twice withEtOH and dried under high vacuum to give2-(3,4-dichloro-benzylidene)-indane-1,3-dione (1c) (1.7 g, 82% yield).

Step b

To a suspension of 2-(3,4-dichloro-benzylidene)-indane-1,3-dione (1c)(1.6 g, 5.2 mmol) in MeOH (13 mL) was added hydrogen peroxide (30%solution, 3 mL). The mixture was cooled to 0° C. and sodium hydroxide (1N, 300 μL) was added dropwise. After addition was completed, stirringwas continued at room temperature for 1 h. The mixture was then pouredinto water (5 mL) and the resulting solid was collected by filtrationand washed with water and MeOH. After drying under high vacuum3-(3,4-dichlorophenyl)-spiro (oxirane-2, 2′-indan)-1′,3′-dione (1d) (1.6g, 95% yield) was obtained.

Step c: Compound 2013

A mixture of 3-(3,4-dichlorophenyl)-spiro(oxirane-2,2′-indan)-1′,3′-dione (1d) (11 g, 33.4 mmol) and 1-benzo(1,3) dioxol-5-yl-pyrrol-2,5-dione (1e) (7.3 g, 33.4 mmol) in toluene(167 mL) was heated to reflux for 16 h. After cooling and concentration,the residue was purified by flash chromatography (SiO₂, gradient 50%EtOAc/hexane to 30% hexane/EtOAc) to give compound 2013 (Table 2)(cis/cis isomer, 17.9 g, 50% yield) and (1f) (trans/cis isomer, 4.1 g,23% yield).

Step d: Compound 1002

To a solution of (2013) (143 mg, 0.27 mmol) in CH₃CN (27 mL) was addedNaOH (0.02N, 135 mL, 0.27 mmol) using a syringe pump over 1 h. After theaddition was completed, the reaction mixture was stirred for an extra 2h and the resulting solution was concentrated and lyophilized to givecompound 1002 (Table 1) (161 mg, 100% yield) as a white solid.

Example 2 Preparation of Compound 4003 (Table 4)

Preparation of Compound (2a)

To a suspension of sodium azide (2.4 g, 36.3 mmol) in CH₃CN (73 mL) wasadded methanesulfonyl chloride (2.8 mL, 36.3 mmol). After stirring for16 h, the reaction mixture was poured over a suspension ofindan-1,3-dione (5.3 g, 36.3 mmol) and cesium carbonate (11.8 g, 36.3mmol) in CH₃CN (50 mL). The mixture was stirred until reaction wascomplete (2 h), and then filtered through Celite. The filter cake waswashed with EtOAc and the organic solvents were removed in vacuo. Theresulting gum was diluted with EtOAc and rinsed successively with NaOH(1 N), H₂O and brine. The organic layer was dried (MgSO₄), evaporatedand the crude oil purified by flash chromatography (SiO₂, 50%hexane/EtOAc) to give 2-diazoindan-1,3-dione (2a) (2.6 g, 42% yield).

Step a

To a mixture of diazoindan-1,3-dione (2a) (317 mg, 1.8 mmol), piperonal(2b) (553 mg, 3.7 mmol) and 1-benzo (1,3) dioxol-5-yl-pyrrol-2,5-dione(1e) (400 mg, 1.8 mmol) in benzene (6 mL) and Rh₂(OAc)₄ (4.5 mg) wereadded and heated to reflux for 1 h. After cooling and concentrating, thereaction mixture was purified by flash chromatography (SiO₂, gradient50% hexane/EtOAc to 30% hexane/EtOAc) to give the desired compound (2c)as beige solid (76.5 mg, 8% yield).

Step b: Compound 4003

Following the same procedure as in Example 1, step d, compound 4003 wasobtained as a white solid.

Example 3 Preparation of Compound 3009 (Table 3)

Compound 2016 was prepared as described in Example 1, steps a to c usingN-(4-acetylphenyl)maleimide in the cycloaddition (step c) to give awhite solid in 40% yield.

Step d

To a solution of 2016 (100 mg, 0.19 mmol) in CH₃CN was added NaOH (0.02N, 9.4 mL, 0.19 mmol) over 1 h using a syringe pump. After addition wascomplete, the solution was concentrated and lyophilized to give a whitesolid which was purified on reverse phase HPLC using a gradient ofCH₃CN/H₂O containing TFA (0.06%). After collecting the desired fractionsand lyophilization compound 3009 was obtained (43 mg, 42% yield) as awhite solid.

Example 4 Preparation of Compound 1022 (Table 1) and Compound 7001(Table 7)

To a solution of 2013 (racemic) (143 mg, 0.27 mmol) in CH₃CN (27 mL) wasadded NaOH (0.02N, 135 mL, 0.27 mmol) using a syringe pump over 1 h.After addition was complete, the reaction mixture was concentrated. Tothe aqueous layer was added HCl (1N, 1 mL) and the resulting acidiclayer was extracted twice with EtOAc. The combined organic layer waswashed with H₂O and brine, dried (MgSO₄) and filtered. To the filtratewas added a solution of CH₂N₂ in Et₂O (excess) and the mixture wasevaporated to dryness. The resulting crude compound was purified bypreparative HPLC using a gradient of CH₃CN/H₂O containing TFA (0.06%).After collecting and lyophilizing the desired fractions, compounds 1022(11 mg, 12% yield), (4a) (9 mg, 10% yield), and 7001 (47 mg, 26% yield)as racemic mixtures were obtained as white solids.

Example 5 Preparation of Compound 8001

Step a

To a solution of 2013 (racemic) (500 mg, 0.93 mmol) in THF/MeOH mixture(20/5 mL) cooled at 0° C. was added NaBH₄ (35 mg, 0.93 mmol). Afterstirring for 30 min, the reaction was quenched with aqueous citric acidsolution (10%) and diluted with EtOAc. The aqueous layer was extractedtwice with EtOAc and the combined organic layers were washed with brine,dried (MgSO₄), filtered and concentrated. Preparative HPLC gave racemicalcohols (5a)+9001 (360 mg, 72% yield, retention times: 14.1 and 12.4min) as well as hydroxylactone (5b) (95 mg, 18% yield, retention time:13.8 min).

The mixture of alcohols was separated by preparative HPLC to generatethe two isomeric compounds as white solids:

Step b

To the mixture of alcohols (5a)+9001 (2:1 ratio, 36 mg, 0.07 mmol) inCH₃CN (10 mL) was added NaOH (0.02N, 3.4 mL, 0.07 mmol) over 2 h using asyringe pump. The CH₃CN was evaporated and the residue was purified bypreparative HPLC. The desired fractions were lyophilized and theresulting solids were retreated with NaOH (1 equiv.) and lyophilized togive compound 8001 (21.3 mg, 59% yield, retention time: 13.7 min) and(5c) (6.0 mg, 17% yield, retention time: 10.9 min) as white solids.

Example 6 Preparation of Compound 5001 (Table 5)

Preparation of Amide (6a)

A mixture of monoethyl fumarate (1 g, 6.9 mmol) and hexachloroacetone(0.5 mL, 3.5 mmol) in CH₂Cl₂ (13 mL) was stirred under nitrogen andcooled to −78° C. Triphenylphosphine (1.8 g, 6.9 mmol) in CH₂Cl₂ (6.9mL) was added dropwise and the mixture was stirred for 20 min. The acylchloride solution was then treated with a solution of3,4-methylenedioxyaniline (946 mg, 6.9 mmol) in CH₂Cl₂ (6.9 mL) followedby Et₃N (0.96 mL, 6.9 mmol) in CH₂Cl₂ (6.9 mL). The reaction mixture wasallowed to warm to room temperature after which the solvent was removedunder high vacuum. The resulting residue was purified by flashchromatography (SiO₂, 20% EtOAc/hexane to give the desired amide (6a)(961 mg, 53% yield) as an orange solid.

Step a

Amide (6a) was reacted with epoxide (1d) using the same procedure as inExample 1, step c to give ester (6b) (77 mg, 14% yield) as an orangesolid.

Step b

Using the same procedure as in Example 1, step d, compound 5001 wasobtained as a white solid (8 mg, 25% yield).

Example 7 Preparation of Compound 4005 (Table 4)

Step a

The same procedure as in Example 1, step a, was followed but usingindan-1,3-dione and trans-cinnamaldehyde (7a) as starting material, togive compound (7b) after purification (11% yield) as an orange solid.

Steps b & c

The same procedures as in Example1, steps b and c were used but usingN-(n-acetylphenyl)maleimide (7d) in the cycloaddition (step c) to affordcompound (7e) (38% yield) as a white solid.

Step d

Hydrolysis was achieved as described in Example 1, step d, to givecompound 4005 (racemic) (50% yield) as a white solid.

Example 8 Preparation of Compound #D1002 (Table 1D)

A: A solution of 8a (9.5 g, 75.4 mmol), malonic acid (15.7 g, 151 mmol)and piperidine (1.3 mL) in pyridine (40 mL) was refluxed overnight. Theresulting mixture was allowed to cool to room temperature whereuponwater (200 mL) was added. The mixture was acidified by the addition ofconcentrated HCl and allowed to stir for 1 h. The mixture was filteredand the solid washed with water. Drying under vacuum gave 8b as a yellowpowder (12.8 g, 100%).

B: To a vigorously stirred solution of 8b (5.9 g, 35 mmol) and 1 N NaOH(46 mL, 46 mmol) in water (40 mL) was added 2% sodium amalgam (82 g, 105mmol) in small portions over 1 h. After complete addition the mixturewas stirred for a further hour. Mercury was removed by decanting and theaqueous solution was acidified with concentrated HCl. Solid NaCl wasadded to saturation and the resulting mixture was extracted with ether.The combined etherial extracts were washed with brine and dried overMgSO₄. Removal of solvent under reduced pressure gave 8c as a brownsolid (3.72 g, 62%).

B: Alternatively, a slurry of 8b (7.5 g, 44.6 mmol) and Pd(OH)₂ (500 mg)in ethanol was stirred under an atmosphere of hydrogen for 18 h.Filtering through glass microfibre and removal of solvent gave 3 as awhite solid (7.0 g, 93%).

C: To a solution of 8c (1.75 g, 10.3 mmol) and oxalyl chloride (1.35 mL,15.4 mmol) in CH₂Cl₂ (50 mL) was added one drop of DMF. The resultingsolution was stirred at room temperature for 2 h. The solvent was thenremoved under reduced pressure and the resulting residue dissolved inCS₂ (50 mL). Solid AlCl₃ (2.05 g, 15.4 mmol) was then introduced and theresulting mixture refluxed overnight. Ice (80 g) was then added followedby concentrated HCl (30 mL) and the resulting mixture was stirred for 30min. Extraction with CH₂Cl₂ was followed by washing with 1N NaOH, brineand drying (MgSO₄). Flash chromatography (20% EtOAc in hexanes) gave 8d(272 mg, 17%) as a yellow solid.

C′: Alternatively, solid 8c (1.0 g, 5.88 mmol) was added in smallportions to warm (75° C.) polyphosphoric acid (8.5 g). Heating wascontinued at 75° C. for one hour after the addition was complete.Cooling to room temperature was followed by dilution with water andextraction with CH₂Cl₂ (3×). The combined organics were dried over MgSO₄and concentrated. Flash chromatography (50% EtOAc in hexanes) gave 8d asa white solid (0.31 g, 35%).

D: A solution of 8d (1.06 g, 6.97 mmol), 3,4-dibromobenzaldehyde (1.84g, 6.97 mmol) and p-toluenesulfonic acid (100 mg) in benzene (25 mL) wasrefluxed for 24 h with azeotropic removal of water. Upon cooling andaddition of ether (25 mL) a solid precipitated which was filtered togive 8e as a tan solid (1.35 g, 49%).

E: To a solution of CrO₃ (50 mg, 0.50 mmol) in CH₂Cl₂ (15 mL) was addedtert-butylhydroperoxide (2.6 mL of a 70% solution in water). Afterstirring for 2 minutes, 8e (1.0 g, 2.51 mmol) was added. After stirringfor 18 h at room temperature the solution was diluted with CH₂Cl₂ andwater and extracted three times with small portions of CH₂Cl₂. Thecombined organics were dried over MgSO₄ and concentrated in vacuo.Trituration of the resulting solid with ether gave 0.61 g (60%) of asolid diketone.

The material so obtained (0.45 g) was dissolved in EtOH (15 mL) to whichwas added 30% H₂O₂ (0.38 mL) and one drop of 1 N NaOH. After stirringfor 3 h the solution was filtered to give 8f as a yellow solid (421 mg,90%).

F: A solution of 8f (0.30 g, 0.70 mmol) andN-[4-(N′-morpholino)phenyl]maleimide (0.18 g, 0.70 mmol) in toluene (8mL) was refluxed for 48 h. After cooling to room temperature a solidformed was filtered and then triturated with THF to provide 8g (75 mg,16%). G: To a solution of 8g (69 mg, 0.10 mmol) in 40% THF:CH₃CN (8 mL)was added NaOH (5.3 mL of a 0.02 M solution in water) over 10 h viasyringe pump. The reaction mixture was purified directly by preparativeHPLC (Chiralcel OD column, isocratic 50% CH₃CN/H₂O, 0.06% TFA) whichafforded enantiomerically pure 8h (2.5 mg, 4%).

Example 9 Preparation of 5-Methyl-6-fluoro-1,3-indanedione

This material is useful as a starting material for preparation ofcompounds of the formula I′ in which R¹ is c-F and b-Me.

A, B: 4-Methylphthalic anhydride 9a (67.5 mmol, 10.94 g) andconcentrated sulfuric acid (10 mL) were placed in a three-neckedround-bottomed flask and the mixture was stirred mechanically at 80° C.A mixture of fuming nitric acid (d=1.5, 4.2 mL) and concentratedsulfuric acid (3.0 mL) was added slowly from a dropping funnel at such arate as to maintain the temperature of the stirred mixture at 100-110°C. Then concentrated nitric acid (d=1.42, 18 mL) was added as rapidly aspossible without causing the temperature to rise above 110° C. Thereaction mixture was heated at 100° C. for two hours, allowed to standat room temperature for 16 h and poured into 30 mL of water. The whiteprecipitate was filtered off and the filtrate was extracted with ethylether. The organic phase was dried with magnesium sulfate, filtered andconcentrated under vacuum. The residual solid (13 g) was dissolved inN,N-dimethylformamide (50 mL) containing potassium carbonate (0.12 mol,16.9 g). Dimethyl sulfate (0.12 mol, 15.4 g, 11.5 mL) was added and themixture was stirred magnetically at room temperature for two hours.N,N-dimethylformamide was evaporated under reduced pressure. The residuewas dissolved in ethyl acetate and the organic phase was washed withwater, brine and dried with magnesium sulfate. The salts were filteredoff and the filtrate was concentrated under vacuum. The residue waspurified by flash chromatography on type H silica gel using hexane/ethylacetate (4/1) as eluent to yield 5.26 g (31%) of dimethyl4-methyl-5-nitrophthalate 9c as a white solid.

C: Dimethyl 4-methyl-5-nitrophthalate 9c (3.41 g, 13.5 mmol) wasdissolved in a mixture of methanol (120 mL) and tetrahydrofuran (20 mL).Palladium hydroxide on carbon (20%, 300 mg) was added and the suspensionwas stirred magnetically at room temperature under hydrogen atmosphere(1 atm) for 16 hours. The reaction mixture was filtered on Celite andthe filtrate was concentrated under vacuum. The residual oil waspurified by flash chromatography on type H silica gel using hexane/ethylacetate (2/1) as eluent, followed by hexane/ethyl acetate (1/1), to give2.88 g (96%) of a colorless oil which corresponded to dimethyl4-methyl-5-aminophthalate 9d.

D: A teflon reactor was charged with dimethyl 4-methyl-5-aminophthalate9d (4.53 g, 20.3 mmol). HF.pyridine (50 mL, ca 1.7 mol HF) was added.The reaction mixture was stirred for 5 min at 0° C. and sodium nitrite(1.55 g, 22.5 mmol) was added to produce a purple solution. The mixturewas stirred for 15 min at room temperature and for 30 min at 120° C. Thereaction mixture was poured onto ice and 4 N sodium hydroxide. Ethylacetate was added and the mixture was filtered. The organic phase wasdried with magnesium sulfate, filtered, washed with 1 M aq HCl, driedagain with magnesium sulfate, filtered and concentrated under vacuum.The residual red liquid was purified by flash chromatography on type Hsilica gel using hexane/ethyl acetate (4/1) as eluent to give 1.54 g(33%) of dimethyl 4-methyl-5-fluorophthalate 9e as a white solid.

E: Dimethyl 4-methyl-5-fluorophthalate 9e (1.65 g, 7.29 mmol) wasdissolved in anhydrous ethyl acetate (4 mL). Sodium hydride (348 mg,14.5 mmol) was added and the mixture was heated at 100° C. for 4 hours.The reaction mixture was cooled to room temperature and a mixturecontaining 10 mL of hexane and 6 mL of ethyl ether/ethanol (1/1) wasadded. The yellow precepitate was triturated for 5 min, filtered anddried under vacuum. The yellow solid (1.07 g) was then suspended in asolution containing water (22 mL) and concentrated hydrochloric acid(2.2 mL) and the suspension was heated for 17 min at 80° C. The mixturewas then lyophilized to give 810 mg (62%) of5-methyl-6-fluoro-1,3-indandione 9f as a beige solid.

Examples of compounds made using 5-methyl-6-fluoro-1,3-indandione arecompounds # A1015 and A1016 (Table 1A).

Example 10 Separation of Mixture to Yield Pure Enantiomers A1006, A1007and A1008 (Table 1A)

Using the same procedure as in Example 1 steps a to d; but starting with5-methyl indan-1,3-dione in step a, and using1-(4-morpholin-4yl-phenyl)pyrrole-2,5-dione in step c, a mixture ofcompounds was obtained which was separated on preparative HPLC using achiral column (Chiracel OD, isocratic eluent 65% CH₃CN/H₂O containing0.06% TFA; UV lamp at 205 nm; flow 7 mL/min.). The resulting threefractions were lyophilized and treated with NaOH (0.02N, 1 equiv.) togive the corresponding sodium salts as white solids.

Compound A1006 was isolated as a mixture of isomers in a 1:1 ratio.

Compounds A1007 and A1008 were each isolated as pure enantiomers.

Example 11 E2-dependent E1 DNA Binding Assay

This assay was modeled on a similar assay for SV40 T Antigen describedby McKay (J. Mol. Biol., 1981,145:471). A 400 bp radiolabeled DNA probe,containing the HPV-11 origin of replication (Chiang et al., 1992, Proc.Natl. Acad. Sci. USA 89:5799) was produced by pcr, using plasmidpBluescript™ SK encoding the origin (nucleotides 7886-61 of the HPV-11genome in unique BAMH1 site) as template and primers flanking theorigin. Radiolabel was incorporated as [³³P]dCTP. Binding assay bufferconsisted of: 20 mM Tris pH 7.6, 100 mM NaCl, 1 mM DTT, 1 mM EDTA.

Other reagents used were protein A-SPA beads (type II, Amersham) and K72rabbit polyclonal antiserum, raised against a peptide corresponding tothe C-terminal 14 amino acids of HPV-11 E1. Following the protocol fromAmersham, one bottle of beads was mixed with 25 mL of binding assaybuffer. For the assay, a saturating amount of K72 antiserum was added tothe beads and the mixture was incubated for 1 h, washed with one volumeof binding assay buffer, and then resuspended in the same volume offresh binding assay buffer. Binding reactions contained 8 ng of E2,approximately 100-200 ng of purified E1, and 0.4 ng of radiolabeledprobe in a total of 80 μL of binding assay buffer. After 1 h at roomtemperature, 25 μL of K72 antibody-SPA bead suspension was added to withthe binding reaction and mixed. After an additional hour of incubationat room temperature, the reactions were centrifuged briefly to pelletthe beads and the extent of complex formation was determined byscintillation counting on a Packard TopCount™. Typically, the signal forreactions containing E1 and E2 was 20-30 fold higher than the backgroundobserved when either E1, E2, or both was omitted.

Example 12 SV40 T Antigen-DNA Binding Assay

Selectivity of the inhibitors according to the invention was assessed byactivity in the E1 or E1-E2-ori binding assays and lack of activity (orlower potency) in the SV40 large T antigen assay.

This assay measures the formation of an SV40 T Antigen (TAg)-origincomplex. The assay was developed by R. D. G. McKay (J. Mol. Biol. (1981)145, 471-488). In principle, it is very similar to the E2-dependentE1-DNA binding assay (Example 12), with TAg replacing E1 and E2, and aradiolabeled SV40 ori probe replacing the HPV ori probe. The assay isused as a counterscreen for the assay of Example 13, since TAg sharesfunctional homology to E1 and E2, but has very low sequence similarity.

The radiolabeled origin-containing DNA probe was made by PCR usingpCH110 plasmid (Pharmacia) as a template. This template encodes the SV40minimal origin of replication at nucleotides 7098-7023. Primers were“sv40-6958sens”=5′-GCC CCT AAC TCC GCC CAT CCC GC (SEQ ID NO. 1), and“sv40-206anti”=5′-ACC AGA CCG CCA CGG CTT ACG GC (SEQ ID NO. 2). The PCRproduct was approximately 370 base pairs long and was radiolabeled using50 μCi/100 μL PCR reaction of dCTP (α-³³P). Subsequent to the PCRreaction, the product was purified using either the Qiagen® PCRpurification kit, or a phenol extraction/ethanol precipitationprocedure. The purified product was diluted to 1.5 ng/μL (estimated bygel electrophoresis) in TE. Fresh preparations had approximately 150,000cpm/μL.

Binding reactions were performed by mixing 30 μl of TAg solution (100ng/well, 200 ng of a ³³P-radiolabeled DNA probe, and 7.5 μl of 10×DNAbinding buffer (200 mM Tris-HCl pH 7.6, 100 mM NaCl, 1 mM EDTA, 10 mMDTT) in a final volume of 75 μl. Binding reactions were allowed toproceed at room temperature for 60 min. The Large T Antigen purchasedfrom Chimerx, at 2.0 mg/mL.

The protein-DNA complexes were immunocaptured using an α-TAg monoclonalantibody (PAb 101, subtype IgG2a, hybridoma obtained from ATCC andantibody purified in-house) bound to protein A-SPA beads.Immunoprecipitation of protein-DNA complexes was carried out for 1 hr atrt. The plates were spun briefly and the precipitated radiolabeled DNAfragments were counted on a TopCount® counter.

Example 13 Cell-based DNA Replication Assay

CHO-K1 cells were transfected using Lipofectamine Plus Reagent(Gibco/BRL) following standard procedure. Cells grown to 40-60%confluence in 100 mm tissue culture dishes were transfectedsimultaneously with 0.5 μg pN9-ORI (HPV-11 minimal origin of DNAreplication), 0.5 μg pCR3-E1 and 0.05 μg pCR3-E2 (containingrespectively HPV-11 E1 full length and HPV-11 E2 full length cloned bythe TA cloning system). After 4 hours of incubation with the DNAmixture, cells were trypsinized, pooled and replated at 20,000cells/well in a 96 well plate. Following 2 hours of attachment at 37°C., serially diluted inhibitor compounds were added for a 2 daysincubation period.

The cells were washed to eliminate the compound, and the total DNA wasextracted using a modified protocol of the QIAamp Blood Kit (QIAGEN).DNA was digested with Hind III (10 U/well) and Dpn1 (20 U/well) for 4hours at 37° C.

Digested DNA (10 μl) was subjected to 23 rounds of PCR amplificationusing the Pwo DNA polymerase Kit (Boehringer Mannheim) modified tocontain 2 U of Pwo DNA polymerase, 10 μCi [α-³³P]dCTP and 2 primers(each at a final concentration of 0.2 μM) per 50 μl reaction.

Cycling consisted of an initial denaturation step at 95° C. for 5 min.,followed by 23 rounds of: denaturation at 94° C. for 30 sec., annealingand extension at 72° C. for 1 min. 30 sec., ending with a finalextension at 72° C. for 10 min. After amplification was completed, 10 μlwas analyzed on 1% agarose gel, subsequently dried at 60° C. for 1 hour,and analyzed by PhosphorImager.

To evaluate the effect of the compound on cellular DNA synthesis (and/orcellular toxicity), cell proliferation ELISA (Boehringer Mannheim),which monitor BrdU incorporation, were performed.

Example 14 Tables of Compounds

All compounds listed in Tables 1 to 10 were found to be active in theE1-E2 DNA assay presented in Example 11 with an IC₅₀ under 50 μM forHPV-11.

Table legend: For IC₅₀ ₂A=50 μM-5 μM; B=5 μM-0.5 μM; C=<0.5 μM

Certain compounds were also tested in the SV40 TAg assay of Example 12and were found to be inactive or less active than in the E1-E2 DNAassay, providing good evidence that these compounds are selectiveagainst the papilloma virus.

In addition, certain compounds were tested in the DNA replicationcellular assay of Example 13. The results obtained indicate that theycan inhibit viral replication.

TABLE 1

racemic, “cis/cis” isomer (form 1) ES MS Table 1 (M + H)⁺ IC₅₀ cpd #R^(4A) R¹ —R⁵ —R³ *(M − H) (μM) 1001 Na — 4-Cl

520  A 1002 Na — 3,4-Cl

552* C 1003 Na — 4-Cl

492* A 1004 Na — 4-Cl

534* A 1005 Na — 4-Cl

516* A 1006 Na — 4-Cl

508* A 1007 Na — 4-Cl

488* A 1008 Na — 4-iPr

526* A 1009 Na — 4-Cl

510* A 1010 Na — 4-Cl

510* A 1011 Na — 4-Cl

474* A 1012 Na — 4-Cl

488* A 1013 Na — 4-Cl

514* A 1014 Na — 4-Cl

542* A 1015 Na — 3-Cl

518* B 1016 Na — 4-CF₃

552* A 1017 CH₃ — 4-Cl

532* A 1018 Na — 3-CH₃

498* A 1019 Na a-F 4-Cl

536* A 1020 Na — 3,5-Cl

552* A 1021 Na — 3,4-Cl

552  C 1022 CH₃ — 3,4-Cl

568  B 1023 Na — 3-OCH₃

515* A 1024 Na — 3,4-CH₃

514  B 1025 Na — 3,4-Cl

566  B 1026 Na — 3,4-F

556  A 1027 Na — 3,4-Br

639* C 1028 Na — 3,4-Cl

657  C 1029 Na — 3-F, 4-Cl

538  B 1030 Na — 3-Cl, 4-F

538  B 1031 Na — 3-CF₃

554  B 1032 Na — 3-Cl

518  B 1033 Na — 3,4-Cl

556  B 1034 Na — 3,4-Cl

595  C 1035 Na — 3,4-Cl

567  C 1036 Na — 3,4-Cl

476  B 1037 Na b-CH₃ 3,4-Cl

568  B 1038 Na — 3,4-Cl

525  B 1039 Na — 4-I

612  B 1040 Na — 3,4-Cl

561  B 1041 Na d-CH₃ 3,4-Cl

568  B 1042 Na a-CH₃ 3,4-Cl

568  B 1043 Na — 3,4-Cl

519  B 1044 Na — 3-Cl

519  A 1045 Na — 3-F, 4-CF₃

572  B 1046 Na — 3,4-Cl

490  B 1047 Na — 3,4-Cl

553  B 1048 Na d-F 3,4-Cl

572  B 1049 Na — 3,4-Cl

600* B 1050 Na — 3,4-Cl

490  B 1051 Na a-F 3,4-Cl

572  B 1052 Na — 3,4-Cl

594  C 1053 Na — 3,4-Cl

588  B 1054 Na — 3,4-Cl

592  C 1055 Na — 3,4-Cl

538  B 1056 Na — 3,4-CH₃

512  B 1057 Na — 3,4-Cl

629  B 1058 Na — 3,4-Cl

550  B 1059 Na — 3,4-F

563  B 1060 Na — 3,4-Cl

566  B 1061 Na — 3,4-F

529  A 1062 Na — 3,4-F

512  A 1063 Na — 3,4-Cl

546  B 1064 Na — 3,4-F

520  A 1065 Na — 3,4-Cl

524  B 1066 Na — 3,4-Cl

550  C 1067 Na — 3-F, 4-CF₃

570  A 1068 Na — 3,4-F

478  A 1069 Na b-Br 3,4-Cl

629  A 1070 Na — 3,4-Cl

534* B 1071 Na — 3,4-CH₃

519* A 1072 Na — 3,4-Br

611  C 1073 Na — 3,4-F

524  A 1074 Na — 3,4-Br

599  C 1075 Na — 3,4-Br

606* B 1076 Na — 3,4-Br

681  C 1077 Na — 3,4-Cl

537* C 1078 Na — 3,4-Br

684  C 1079 Na — 3,4-Br

673 (M + 18) A 1080 Na — 3-CN

511  A 1081 Na — 3,4-Br

646  C 1082 Na — 3,4-Cl

516  B 1083 Na — 3,4-F

562  B 1084 Na — 3,4-Br

644  C 1085 Na — 3-CN

552  A 1086 Na — 3,4-Br

676  C 1087 Na —

558  A 1088 Na — 3,4-Br

639  B 1089 Na — 3,4-Br

639  C 1090 Na

3,4-Cl

602  A 1091 Na

3,4-Cl

602  A 1092 Na — 3,4-Br

  654.8* C 1093 Na — 3-Cl, 4-F

503  A 1094 Na — 3-Cl, 4-F

536  B 1095 Na

3,4-Cl

602  A 1096 Na — 3,4-Cl

546  B 1097 Na — 3,4-Br

 627.9 C 1098 Na — 3,4-Cl

550  B 1099 Na — 3,4-Br

606  C 1100 Na — 3,4-Cl

614  B 1101 Na — 3,4-Cl

544  B 1102 Na — 3,4-Br

709  B 1103 Na — 3,4-Br

 701.9 B 1104 Na — 3,4-Cl

 623.9 B 1105 Na — 3,4-Br

 635.9 B 1106 Na b-F 3,4-Cl

570  B 1107 Na c-F 3,4-Cl

570  B 1108 Na — 3,4-Cl

565* B 1109 Na — 3,4-Br

336  B 1110 Na — 3,4-Br

634  C 1111 Na — 3,4-Cl

546  B 1112 Na — 3,4-Cl

594  B 1113 Na — 3,4-Br

638* C 1114 Na c-Cl 3,4-Cl

586  B 1115 Na — 3-Cl, 4-F

522  B 1116 Na b-Cl 3,4-Cl

586  A 1117 Na — 3,4-Cl

599  B 1118 Na — 3,4-Br

684  B 1119 Na — 3,4-Br

634  C 1120 Na — 3-Cl, 4-F

530  B 1121 Na — 3-Cl, 4-F

608  B 1122 Na — 3-Cl, 4-F

530  A 1123 Na — 3,4-Cl

544  C 1124 Na — 3,4-Cl

540  B 1125 Na — 3,4-Cl

528  C 1126 Na — 3,4-Cl

538  B 1127 Na — 3,4-Cl

535  B 1128 Na — 3,4-Cl

590  B 1129 Na c-OMe 3,4-Cl

 581.9 A 1130 Na b-OMe 3,4-Cl

  579.9* A 1131 Na — 3-Cl, 4-F

578  B 1132 Na — 3,4-F

492  A 1133 Na — 3,4-Cl

536* B 1134 Na — 3,4-Br

  665.6* C 1135 Na — 3,4-Cl

570* C 1136 Na — 3,4-Cl

554  A 1137 Na — 3,4-Cl

598  B 1138 Na — 3,4-Cl

544  C 1139 Na — 3,4-Cl

594  B 1140 Na — 3,4-Cl

830  B 1141 Na — 3-NHC(O)(CH₂)₃CH₃, 4-Cl

617  B 1142 Na — 3,5-Cl

590  C 1143 Na b-F 3,4-Br

 702.9 C 1144 Na c-F 3,4-Br

 702.9 C

TABLE 1A

enantiomerically pure, “cis/cis” isomer (form 1) Table ES MS 1A (M + H)⁺IC₅₀ cpd # R^(4A) R¹ —R⁵ —R³ *(M − H) (μM) A1001 Na — 3,4-Br

  683.8 A A1002 Na — 3,4-Br

  683.8 C A1003 Na mixture b-Me & c-Me 3,4-Cl

568 A A1004 Na b-Me 3,4-Cl

568 A A1005 Na c-Me 3,4-Cl

568 C A1006 Na mixture b-Me & c-Me 3,4-Cl

608 A A1007 Na b-Me 3,4-Cl

608 B A1008 Na c-Me 3,4-Cl

608 C A1009 Na mixture b-Me & c-Me 3,4-Br

  697.9 A A1010 Na b-Me 3,4-Br

  697.9 B A1011 Na c-Me 3,4-Br

  697.9 C A1012 Na — 3,4-Br

 683* A A1013 Na — 3,4-Br

 683* C A1014 Na c-Me 3,4-Br

699 C A1015 Na b-F, c-Me 3,4-Br

  716.8 C A1016 Na b-Me, c-F 3,4-Br

717 B

TABLE 1B

enantiomerically pure, “cis/cis” isomer (form 3) ES MS Table 1B (M + H)⁺IC₅₀ cpd # R¹ R⁵ R³ *(M − H) (μM) B1001 b-Me, c-Me (mixture) 3,4-Br

699 A B1002 b-Me 3,4-Br

699 C B1003 c-Me 3,4-Br

699 C B1004 b-Me 3,4-Br

676 A B1005 c-Me 3,4-Br

676 A B1006 b-Me 3,4-Br

676 B B1007 c-Me 3,4-Br

676 C B1008 b-F, c-Me 3,4-Br

716.6 C

TABLE 1C

ES MS Table 1C (M + H)⁺ IC₅₀ cpd # R⁵ R³ *(M − H) (μM) C1001 3,4-Cl

572 A C1002 3,4-Br

704 B C1003 3,4-Br

747 B

TABLE 1D

enantiomerically pure “cis/cis” isomer (form 3) ES MS Table 1D (M + H)⁺IC₅₀ cpd # R⁵ R³ *(M − H) (μM) D1001 3,4-Cl

572 C D1002 3,4-Br

704 C D1003 3,4-Br

747 C D1004 3,4-Br

691 C

TABLE 2

racemic, “cis/cis” isomer (form 2) ES MS Table 2 (M + H)⁺ IC₅₀ cpd # —R⁵—R³ *(M − H) (μM) 2001 4-Cl

500* A 2002 4-Cl

516* A 2003 4-Cl

498* A 2004 4-Cl

474* A 2005 3-Cl

500* B 2006 4-Cl

470* A 2007 4-Cl

490* A 2008 4-CF₃

534* A 2009 4-Cl

490* A 2010 4-Cl

470* A 2011 4-Cl

524* A 2012 4-Cl

496* A 2013 3,4-Cl

534* B 2014 3-CH₃

480* A 2015 4-Cl

524* A 2016 3,4-Cl

552  (M + 18) B 2017 4-I

612  (M + 18) A 2018 3,4-Cl

624  (M + 18) A 2019 3,4-Cl

524  (M + 18) B 2020 4-OH, 5-Cl

534  (M + 18) B 2021 3,4-Cl

526  (M + 18) C 2022 3,4-Cl

827  A 2023 3,4-Br

667  C

TABLE 3

racemic, “cis/cis” isomer (form 3) ES MS Table 3 (M + H)⁺ IC₅₀ cpd # R¹—Y —R³ *(M − H) (μM) 3001 —

 552* B 3002 —

482 A 3003 —

 488* A 3004 —

 566* A 3005 —

 518* A 3006 —

510 A 3007 —

538 A 3008 —

637 A 3009 —

552 C 3100 —

540 A 3011 —

526 B 3012 —

 569* B 3013 c-Me

697 C 3014 —

588 B 3015 —

544 B 3016 b-F

702 C 3017 c-F

702 C

TABLE 4

racemic, “cis/cis” isomer (form 1) ES MS Table 4 (M + H)⁺ IC₅₀ cpd # —Y—R³ *(M − H) (μM) 4001

536 B 4002

526 A 4003

530 A 4004

534 B 4005

510 A 4006

588 A 4007

588 B 4008

523 A 4009

523 A 4010

 576* B 4011

 522* A 4012

589 A

TABLE 5

racemic, trans/trans isomer^(‡) (form 1) Table 5 ES MS IC₅₀ cpd # —R⁵—R³ (M + H) (μM) 5001 3,4-Cl

554 B ^(‡)relative stereochemistry shown

TABLE 6

racemic, trans/cis isomer^(‡) (form 1) Table 6 ES MS IC₅₀ cpd # —R⁵ —R³(M + H)⁺ (μM) 6001 3,4-Cl

568 A ^(‡)relative stereochemistry shown

TABLE 7

racemic, “cis/cis” isomer^(‡) (form 1) Table 7 ES MS IC₅₀ cpd # R^(4A)—R⁵ —R³ (M + H)⁺ (μM) 7001 OCH₃ 3,4-Cl

582 A ^(‡)relative stereochemistry shown

TABLE 8

racemic, “cis/cis” isomer^(‡) (form 2) Table 8 ES MS IC₅₀ cpd # —R⁵ —R³(M + H)⁺ (μM) 8001 3,4-Cl

556 B ^(‡)relative stereochemistry shown

TABLE 9

racemic, “cis/cis” isomer^(‡) (form 2) Table 9 ES MS IC₅₀ cpd # W —R⁵—R³ (M + H)⁺ (μM) 9001

3,4-Cl

540 A ^(‡)relative stereochemistry shown

TABLE 10

racemic, cis/trans isomer^(‡) (form 1) Table 10 ES MS IC₅₀ Cpd # —Y —R³(M + H)⁺ (μM) 10,001

683 B ^(‡)relative stereochemistry shown

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer sequence <400> SEQUENCE: 1gcccctaact ccgcccatcc cgc            #                  #                23 <210> SEQ ID NO 2 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer sequence <400> SEQUENCE: 2accagaccgc cacggcttac ggc            #                  #                23

What is claimed is:
 1. A compound of formula (I), or an enantiomer ordiastereoisomer thereof:

wherein: A is a 5- or 6-membered carbocyclic ring; X is H and W is OH;or X and W together form a carbonyl group or an epoxide; R¹ is H; or oneor two substituents independently selected from the group consisting of:hydroxy; halo; lower alkyl; lower alkoxy; lower thioalkyl; haloalkyl(e.g. trifluoromethyl); or —C(O)R² wherein R² is lower alkyl, aryloxy orbenzyloxy; Y is phenyl optionally mono- or di-substituted with R⁵ orC(O)R⁶, wherein R⁵ is lower alkyl, lower cycloalkyl, lower alkoxy, halo,hydroxy, nitrile or trifluoromethyl, and R⁶ is lower alkyl, lowercycloalkyl, lower alkoxy, hydroxy or trifluoromethyl; said phenyl ringbeing optionally fused with a saturated or unsaturated 4 to 6-memberedcarbocyclic ring; or Y is ethylene-phenyl, said ethylene moiety beingoptionally mono-substituted with lower alkyl, wherein said phenyl ringis optionally mono- or di-substituted with R⁵ or C(O)R⁶, wherein R⁵ andR⁶ are as defined above; said phenyl ring being optionally fused with asaturated or unsaturated 4 to 6-membered carbocyclic ring; R³ isselected from the group consisting of: aryl, mono- or di-substitutedwith: morphiline, said morpholine optionally mono- or di-substitutedwith lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy,nitrile, trifluoromethyl, C(O)R⁶ wherein R⁶ is as defined above; and R⁴is a carboxylic acid, a salt or an ester thereof.
 2. A compound selectedfrom:

wherein A, X, R¹, Y, R³, and R⁴ are as defined in claim
 1. 3. A mixtureof compound I(a) and compound I(b), according to claim
 2. 4. A mixtureof compound I(c) and compound I(d), according to claim
 2. 5. A compoundmixture according to claim 3, wherein said mixture is racemic.
 6. Acompound mixture according to claim 4, wherein said mixture is racemic.7. A compound I(a) according to claim 2, as a pure enantiomer.
 8. Acompound I(c) according to claim 2, as a pure enantiomer.
 9. A compoundaccording to claim 1 wherein X is H and W is OH; or X and W form acarbonyl group.
 10. A compound according to claim 9 wherein X and W forma carbonyl group.
 11. A compound according to claim 1 wherein ring A isa benzene ring, as represented by the formula I′:

wherein X, R¹, W, Y, R³, and R⁴ are as defined in claim
 1. 12. Acompound according to claim 1, wherein R¹ is H; or one or twosubstituents independently selected from the group consisting of:hydroxy; halo; lower alkyl; lower alkoxy; lower thioalkyl; haloalkyl; or—C(O)R² wherein R² is lower alkyl, aryloxy or benzyloxy.
 13. A compoundaccording to claim 12, wherein R¹ is H, halo or C₁₋₄ alkyl.
 14. Acompound according to claim 13, wherein R¹ is H, fluoro or methyl.
 15. Acompound according to claim 14, wherein R¹ is H or methyl.
 16. Acompound according to claim 1, wherein Y is phenyl optionally mono- ordi-substituted with R⁵ or C(O)R⁶, wherein R⁵ is lower alkyl, lowercycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, andR⁶ is lower alkyl, lower cycloalkyl, lower alkoxy, hydroxy ortrifluoromethyl; said phenyl ring being optionally fused with asaturated or unsaturated 4 to 6-membered carbocyclic ring; or Y isethylene-phenyl, said ethylene moiety being optionally mono-substitutedwith lower alkyl, wherein said phenyl ring is optionally mono- ordi-substituted with R⁵ or C(O)R⁶, wherein R⁵ and R⁶ are as definedabove; said phenyl ring being optionally fused with a saturated orunsaturated 4- to 6-membered carbocyclic ring.
 17. A compound accordingto claim 16, wherein Y is naphthyl, CH═CH-phenyl, C(CH₃)═CH-phenyl orphenyl, wherein the phenyl ring is optionally mono- or di-substituted atthe 3, 4, or 5 position with R⁵, wherein R⁵ is halo, C₁₋₄ alkyl, hydroxy, CF₃ or NHC(O)-(lower alkyl).
 18. A compound according to claim 17,wherein Y is phenyl optionally substituted with: 3,4-Cl; 3-F, 4-Cl;3-Cl, 4-F; 3,4-Br; 3-F,4-CH₃; 3,4-CH₃; 3-CF₃ or; NHC(O)—(CH₂)₃CH₃.
 19. Acompound according to claim 18, wherein Y is phenyl optionallysubstituted with: 3,4-Cl or 3,4-Br.
 20. A compound according to claim 1,wherein R³ is: (C₁₋₆ alkyl)phenyl wherein the phenyl ring is optionallysubstituted with: morpholine, said morpholine optionally mono- ordi-substituted with lower alkyl, lower alkoxy, halo, hydroxy, nitrile ortrifluoromethyl.
 21. A compound according to claim 20, wherein R³ is:


22. A compound selected from the group consisting of: compounds havingthe following formula:

wherein R^(4A), R¹, R⁵ and R³ are as defined as follows: Cpd # R^(4A) R¹—R⁵ —R³ 1034 Na — 3,4-Cl

1059 Na — 3,4-F

1078 Na — 3,4-Br

1085 Na — 3-CN

1128 Na — 3,4-Cl

1143 Na b-F 3,4-Br

1144 Na c-F 3,4-Br


23. A compound selected from the group consisting of: compounds havingthe following formula:

wherein R^(4A), R¹, R⁵, and R³ are as defined as follows: Cpd # R^(4A)R¹ —R⁵ —R³ A1012 Na — 3,4- Br

A1013 Na — 3,4- Br

A1014 Na c-Me 3,4- Br

A1015 Na b-F, c-Me 3,4- Br

A1016 Na b-Me, c-F 3,4- Br


24. A compound selected from the group consisting of: compounds havingthe following formula:

wherein R¹, R⁵, and R³ are as defined as follows: Cpd # R¹ R⁵ R³ B1001b-Me, c-Me (mixture) 3,4-Br

B1002 b-Me 3,4-Br

B1003 c-Me 3,4-Br

B1008 b-F, c-Me 3,4-Br


25. A compound selected from the group consisting of: compounds havingthe following formula:

wherein R⁵ and R³ are as defined as follows: Cpd # —R⁵ —R³ 2023 3,4-Br


26. A compound selected from the group consisting of: compounds havingthe following formula:

wherein R¹, Y, and R^(3 are as defined as follows:) Cpd # R¹ —Y —R³ 3016b-F

; and 3017 c-F

.


27. A compound having the following formula:

wherein Y and R³ are as defined as follows: Cpd # —Y —R³ 10,001


28. A pharmaceutical composition comprising an anti-papillomavirusvirally effective amount of a compound of formula (I), according toclaim 1, or a therapeutically acceptable salt or ester thereof, inadmixture with a pharmaceutically acceptable carrier medium or auxiliaryagent.
 29. A method for treating a papillomavirus viral infection in amammal by administering to the mammal an anti-papilloma virus virallyeffective amount of a compound of formula (I), according to claim 1, ora therapeutically acceptable salt or ester thereof, or a pharmaceuticalcomposition comprising an anti-papillomavirus virally effective amountof a compound of formula (I) according to claim 1, or a therapeuticallyacceptable salt or ester thereof, in admixture with a pharmaceuticallyacceptable carrier medium or auxiliary agent.
 30. A method forinhibiting the replication of papillomavirus by exposing the virus to anamount of a compound of formula (I), according to claim 1 inhibiting thepapilloma virus E1-E2-DNA complex, or a therapeutically acceptable saltor ester thereof, or a composition comprising an anti-papillomavirusvirally effective amount of a compound of formula (I) according to claim1, or a therapeutically acceptable salt or ester thereof, in admixturewith a pharmaceutically acceptable carrier medium or auxiliary agent.31. A method of preventing perinatal transmission of HPV from mother tobaby, by administering a compound of formula (I), according to claim 1,to the mother prior to giving birth.
 32. A compound I(b) according toclaim 2, as a pure enantiomer.
 33. A compound I(d) according to claim 2,as a pure enantiomer.